Immunomodulatory potential of Sarcophaga argyostoma larval hemolymph as a natural alternative to berenil in treating Trypanosoma evansi in vivo

This study compared effects of diminazene aceturate (berenil), commonly used to treat domestic animals infected with Trypanosoma evansi, with the hemolymph of Sarcophaga argyostoma larva. The hemolymph may be acting as a possible natural alternative to berenil, based on immunomodulation mediated inflammatory response. Inflammatory mediators and histopathological changes in liver, kidney, and spleen of albino mice experimentally infected with T. evansi were studied. Mice were divided into five groups: G1, uninfected, untreated (negative control); G2, T. evansi infected (positive control); G3, infected and treated with berenil; G4, infected and treated with hemolymph; G5, infected and treated with hemolymph 3 days before infection (prophylactic group). Animals in (G4) and (G5) exhibited a significant overall reduction in serum levels of IFN-γ. However, the reduction in TNF-α and IL-6 levels was more limited compared to (G2) and (G3). Notably, an elevation in IL-10 levels was observed compared to animals in other groups. Furthermore, the groups treated with hemolymph demonstrated an alleviation of T. evansi infection in contrast to the other groups. This study highlights that the administration of Sarcophaga argyostoma larval hemolymph at a dosage of 0.5 ml/kg significantly inhibited T. evansi organisms in vivo, showcasing a pronounced trypanocidal effect.


Preparation of hemolymph of Sarcophaga argyrostoma larva
Following CO2 anesthesia, the anterior tips of 50 larvae per sample were clipped with sterile, fine scissors to utilize the hemolymph, and then placed in an ice-cold Eppendorf with a few crystals of phenylthiourea to block the melanization process 24 .The hemocytes and hemolymph were separated by centrifugation for 10 min at 1000g and 4 °C25 .The supernatant was then stored at − 20 °C until it was needed.

Gas-liquid chromatography (GLC) analysis for carbohydrates
Chromatographic analysis using GLC was performed by the Thermo system (Ultimate 3000) to analyze and detect carbohydrates, phenolics and fatty acids.The system comprised a pump, automatic sample injector, and a DELL-compatible computer running the Cromelion7 interpretation program.Detection was carried out using a RefractoMax520 refractive index detector.An aminopropyl-bonded phase column (4 gm high-performance carbohydrate column, waters) running at 30º C was used for the analysis.An isocratic solution of acetonitrile and water (75:25) made up the mobile phase.To reduce interference from NaCl, sodium chloride was added at a concentration of 0.125% (w/v) 26 .

Gas-liquid chromatography (GLC) analysis for phenolics
A diode array detector (DAD-3000) was used to measure phenolics.The Thermo-hypersil reversed phase C18 column, measuring 2.5 × 30 cm, operated at 25 °C.The mobile phase comprised 0.05% trifluoroacetic acid/acetonitrile (solvent A) and distilled water (solvent B).UV absorption spectra for both standards and samples were collected within the 230-400 nm range.Prior to analysis, the mobile phase, standard solutions, and samples were degassed and filtered through a Millipore 0.45 µm membrane filter.Compound identification was achieved by comparing their retention times and UV absorption spectra to those of the standards 27 .

Gas-liquid chromatography (GLC) analysis for fatty acids
The extracted fatty acids from both the sample and the standards were converted into the corresponding methyl esters using an ethereal diazomethane solution.Subsequently, the methyl esters of the fatty acids were analyzed using a GCV Pye-Unicam gas chromatography equipment.The proportions of the methyl esters of fatty acids were determined using a GLC column 28 .

Preparation and identification of amino acids by using amino acid analyzer
The SYKAM high-performance analyzer was employed for amino acid analysis and identification.Defatted hemolymph solution was obtained by three cycles of boiling under reflux with 50 ml of 50% ethanol, yielding one milliliter each time over three hours.To clarify the mixed ethanolic solutions, they were filtered and treated with a 10% trichloroacetic acid solution.The resulting supernatant was concentrated to 5 ml under reduced pressure and cleansed with distilled water.The filtrate was adjusted to a volume of 100 ml using distilled water.Five milliliters of the diluted sample were vacuum-dried at 70 °C and subsequently dissolved in a five-milliliter loading buffer (pH 2; 0.2 N sodium citrate buffer).After filtration through a 0.45-micron pore filter, the substance was introduced into the amino acid analyzer 29 .
Peaks were identified by comparing the relative retention times of each compound with those of reference materials.Following Farag's et al. 29 method, the ratio of each compound's partial area to its total area was utilized to estimate their respective proportions.

Trypanosoma evansi isolate
T. evansi was originally isolated from a camel naturally infected and raised in a rural area in the Assuit province in upper Egypt (27° 11′ N 31° 10′ E).Thin blood films were prepared from ear vein, dried, fixed in absolute methyl alcohol and stained with Giemsa stain according to Soulsby 30.

Experimental animals
Forty male albino mice (Mus domesticus) with average weight 35-40 g were obtained from Veterinary Collage, Assuit University, Egypt.Animals were examined and acclimated for 10 days before use.The animals were housed in standard laboratory conditions, maintained at 21°C with 16% moisture, provided ad-libitum access to water, and fed a diet consisting of 20% protein, 3% fat, and 22% fiber.Following experimental infection with T. evansi, blood samples were taken daily from day 4 to day 10 until the animals were sacrificed.
Group 4 (G4): Infected and treated with a single dose of 0.5 ml/kg hemolymph upon detection of the parasite in the blood.

Trypanosoma evansi inoculation
All animals in G2, G3, G4, and G5 were inoculated intraperitoneally with 0.3 ml of fresh blood, obtained from camels previously infected with T. evansi.The dose was approximately 10 4 parasites/animal.

Berenil preparation and administration
Drugs were dissolved and prepared as aliquots for intraperitoneal (I.P.) injection, following manufacturer's instructions.The dose of diminazene aceturate (Berenil, Hoechst, Germany) was 0.5 ml/kg of body weight according to MSD animal health instructions, 2020.

Daily follow up and sampling
Daily examination in mice was checked.1st, 2nd, and 3rd days no trypanosomes were appeared in the blood of infected mice (First three days considers prepatent period), the detection of parasitemia and counting number of trypanosomes was recorded from 4th day until sacrifice at the end of the experiment (on day 10 post infection) by cutting the tip of tail and collecting a drop of blood on clean glass slides.Blood was then examined under a microscope at 100 × according to Pizzi-Brener 31 .Trypanosomes in thin blood film were counted according to Kolmer method 32 .Upon completion of the experiment, blood was collected from the ocular vein, and serum was prepared for cytokines analysis.Animals were dissected immediately for histopathology preparation.

Histopathological preparation and analysis
Samples of liver, kidney, and spleen were washed in saline solution and fixed in 10% formalin for 24-48 h at room temperature, embedded in paraffin, and cut into 4-6 µm sections.Sections were stained with hematoxylin-eosin stain using standard histological techniques 33 .Histopathological examination of tissues uses an Olympus BX51 light microscope.

Statistical analysis
Data were analyzed using the descriptive statistics procedure in the SPSS (IBM, USA, v23) statistical package.The difference between means was evaluated by one-way analysis of variance (ANOVA) followed by post hoc test (LSD).Means values were calculated for all treatments.The least significant difference method was used for comparing means.Differences among means with p < 0.05 were accepted as statistically significant, and differences of 0.05 < p < 0.10 were accepted as tendencies.

Determination of chemical compounds using gas-liquid chromatography (GLC) and amino acid analyzer in the hemolymph of Sarcophaga argyostoma larva
The chemical compounds, including carbohydrates, phenolics, and fatty acids, are listed in Table 1, with their identification based on parameters such as relative time (minutes), area (mAU*min), height (mAU), relative area (%), relative height (%), and amount (µg).The results indicate the presence of lactose and fructose, classified as carbohydrates (sugars), along with 5 phenolic compounds and 21 fatty acids.Additionally, the amino acid analysis in Table 2 revealed the presence of 17 compounds identified by retention time (min.),response amount (PPM), and amount (%).

Parasitemia estimation
The hemolymph of Sarcophaga argyrostoma larva was investigated as an a trypanocidal alternative for berenil.The concentration in this experiment was 10 mg/ml, and dose rate was 0.5 ml/kg BW.The average prepatent period ranged from 2-3 days.Trypanosomes are detected within all groups with average incidence/mm 3 32 × 10 4 in G2, 30 × 10 4 in G3, 29 × 10 4 in G4 and 29 × 10 4 in G5.All treated mice became All treated mice became aparasitemic at the end of the experiment with a clearance percentage of 100%.The time courses of parasitemia show initial clearance of parasites began on day six in G4 and day five in G5 post-infection, while in G3 mice, treated with berenil, parasites in blood decreased slowly before finally disappearing on day eight post-infection (Fig. 1).

Inflammatory mediators
Immunomodulation of inflammatory cytokine levels in different treated, infected, and negative control after 10 days of infection showed in Table 3. Serum levels of IFN-γ decreased (P < 0.001) in G3, G4, and G5-treated groups respectively compared to both positive and negative control groups.An increase in the serum levels of   www.nature.com/scientificreports/TNF-α and IL-6 in infected and treated animals was observed compared to the -ve control group.In contrast, IL-10 serum level was reduced in G3 mice compared to the -ve control group but elevated in G4 and G5 compared to the + ve and −ve control group respectively (Table 3).

Histopathological findings
Liver Liver variably changes from moderate histopathological changes in G3 mice to severe degenerative changes G2 are shown in Fig. 2. Livers from mice in G2 that did not receive treatment showed hepatomegaly, diffuse and massive degenerative changes, including granular cytoplasm and pyknotic or vesicular nuclei (Fig. 2A).
Significant congestion in central vein with massive, degenerated hepatocytes was also present (Fig. 2B).Livers from mice in G3, treated with berenil, showed dilatation of hepatoportal blood vessels and diffuse vacuolation of hepatic parenchyma (Fig. 2C).Livers of mice in G4 and G5, treated with hemolymph after and before infection respectively, maintained their original structure (Fig. 2D,E) as also observed in livers normal negative control mice (G1) (Fig. 2F).

Kidney
The kidney histopathological changes are shown in Fig. 3. Mice in G2 showed multifocal aggregation of mononuclear cells and degeneration in both renal tubules and glomeruli (Fig. 3A), severe congestion in interstitial blood vessels and thickened muscular walls (Fig. 3B).Kidneys of mice from G3 showed congestion in interstitial blood vessels with degenerated and disintegrated renal tubules (Fig. 3C).The kidneys of mice in G4 and G5 retained nearly normal architecture (Fig. 3D,E) when compared with the negative control group (G1) (Fig. 3F).

Spleen
The main histopathological features in the spleen are shown in Fig. 4. The spleen of the infected group (G2) consisted of necrosis in splenic lymphoid follicles (Fig. 4A) and atrophy in splenic lymphoid follicles in mice in G3 (Fig. 4B).Upon treatment with extract, spleen structure remained almost normal.The extract significantly spared histological features of the spleen (Fig. 4C,D).The spleen of negative normal control is shown in Fig. 4E.

Discussion
Several discussions of natural insect products and their potential for development into drugs to treat T. evansi diseases are available.Propolis extract can prolong the life of rats infected with T. evansi 34 and methanol extracts of Musca domestica larva display trypanocidal properties 35 .The use of Sarcophage larvae for treatment of certain bacterial infections is years old.Okada and Natori 36 purified three antibacterial proteins from the hemolymph of Sarcophaga larvae.In this study chromatographic results showed that the hemolymph of S. argyostoma larvae is a valuable resource rich in phenolic, fatty, and amino acids.The presence of phenolic content in the hemolymph was identified and quantified by the analytical method of Gas-Liquid Chromatography (GLC).The extracts revealed the presence of phenolic acids such as quinic acid, apigenin, chlorgenic acid, gallic acid and cinnamic acid.Júnior et al. 37 detected quinic acid, chlorogenic acid, and gallic acid in the leaf extract of Guazuma ulmifolia, a natural product that exhibits significant antiparasitic properties.Additionally, according to other study findings, specific phenolic substances in plant extracts possess the ability to inhibit kinetoplastid species such as Leishmania and Trypanosoma 38 .
As per the analysis of fatty acids, the chromatographic findings revealed the presence of 21 fatty acids.Hirazawa et al. 39 suggested that short and medium-chain fatty acids (C4-C10) such as butyric acid, caproic acid, enanthic acid, and caprylic acid exhibit antiparasitic effects against Cryptocaryon irritans ciliates.Additionally, Goodman and McFadden 40 demonstrated that synthesis inhibitors incorporating fatty acids offer a promising avenue for developing new anti-parasitic compounds.
One of the primary causes of insects' remarkable capacity to adapt to survive is the fact that antimicrobial peptides are mostly produced in the fat bodies and blood cells of insects 41 .Our study revealed the presence of 17 amino acids that belong to antimicrobial peptides (AMPs), found in amino acid-rich species 42 , such as glycine, Table 3.The change of inflammatory mediator' levels (pg/ml) in different infected and treated groups compared with control at the end of experiment.Values are expressed as mean ± standard deviation (SD), n = 8 mice.G1 negative control, G2 T. evansi infected, G3 berenil-treated, G4 hemolymph-treated group, G5 hemolymph-prophylactic group, IFN-γ interferon-gamma, TNF-α tumor necrosis factor, IL-6 interleukin-6, IL-10 interleukin-10.*P < 0.05; **P < 0.01; ***P < 0.001 vs. negative control group.a P < 0.05 vs. positive control group (T.evansi infected group).b P < 0.05 vs. berenil-treated group.arginine, proline, and histidine.Antiparasitic peptides demonstrate their ability to combat diseases like malaria and leishmaniasis 43 .Specific peptides, including lysine, aspartic acid, glutamic acid, and leucine derived from bee royal jelly, have exhibited considerable efficacy against the Leishmania parasite 44 .
Common problems with the use of berenil for the treatment of T. evansi infection are toxicity at therapeutic doses, including tachycardia and CNS signs such as ataxia, nystagmus, and opisthotonos 45 .Parasitemia significantly increased in G2 mice (positive control) while significantly reduced in the mice treated with berenil and hemolymph.The main difference between these two treatments is that mice treated with berenil became aparasitemic on the 8th day after infection, while mice treated with hemolymph were aparasitemic on the 5th day post-infection.These results agree with the previous study of Shittu et al. 35 , who recorded a significant decrease in T. brucei parasitemia in mice treated with 400 mg/kg of a methanolic extract of Musca domestica larvae.
The three primary proinflammatory cytokines associated with parasite infection-IFN-γ, TNF-α, and IL-6have been identified.Research has shown that the anti-inflammatory cytokine IL-10 functions to counteract the effects of these proinflammatory cytokines, contributing to the maintenance of immunological homeostasis and regulation of inflammatory responses.In this study, IFN-γ showed a reduction in extract-treated groups compared to all different groups, also TNF-α and IL-6 significantly decreased in comparison with other treated and infected animals, IL-10 showed higher values than other different groups.IFN-γ plays a crucial function in identifying and getting rid of parasites by mediating host-pathogen interactions.The initiation of a cascade of pro-inflammatory responses is caused by a complex interplay between immune cell activity and IFN-γ through coordinated integration of signals from other pathways involving cytokines and Pattern Recognition Receptors (PRRs) such as Interleukin (IL)-4, Lipopolysaccharide (LPS), TNF-α, IL-6 as in trypanosomiasis 46 .
INF-γ levels in the serum may be associated with resistance to parasitemia and regulation of the condition by activating macrophages, which eliminates the parasite from the bloodstream.Several authors have described this pathway for trypanosomiasis [47][48][49] .
TNF-α exhibits trypanolytic properties linked to elevated levels during trypanosomiasis infection 50,51 .This elevation potentially aids in controlling parasitemia, as observed by a subsequent decrease in bloodstream parasites following increased TNF-α levels.The inability to effectively manage parasitemia was associated with excessive production of proinflammatory cytokines, including IFN-γ and TNF-α, due to impaired activation of alternatively activated macrophages 52 .While the production of IFN-γ and TNF-α is crucial for protection, excessive production is detrimental, rendering infected mice more susceptible and leading to their death 53 .An increase in IL-6 was also described in mice infected with T. brucei 54 .IL-6 deficient was more susceptible to T. cruzi, infection and increased mortality rates 55 .www.nature.com/scientificreports/IL-10 has been shown to have anti-inflammatory properties to protect hosts from a number of parasitic diseases, such as trypanosomiasis 56 .IL-10 acts to downregulate excessive effector activities of both T cells and macrophages 53 that are linked to protection.Sternberg et al. 54 (2005) and Baldissera et al. 45 reported that infection with T. brucei in a murine model showed upregulation of IL-10 levels and showed only mild inflammatory pathology.This finding is consistent with the present results in mice treated with hemolymph that showed no pathological changes.
In fact, protection during infection with trypanosomes (lower parasitemia and prolonged survival) is associated with the ability to switch from Th1 to Th2 response during the later stages of infection 52 .In line with this, the reduction of IFN-γ, TNF-α, and IL-6 signaling (which are key cytokines that drives Th1 differentiation), and elevation of IL-10 (which is a key cytokine that drives Th2 differentiation) that observed in hemolymph-treated trypanosomiasis mice of this study, led to the ability to control parasitemia compared to other counterpart mice.
During the initial examination of organs, hepatomegaly, observed in this study, is compatible with Brown and Losos 57 .The most drastic changes in livers were recorded in G2, followed by G3.G2 mouse livers showed massive degenerative changes in hepatocyte nuclei and cytoplasm possibly caused by consumption of oxygen by proliferating trypanosomes.Such consumption could deprive tissues of oxygen, resulting in degenerative changes 58 .The congestion and dilation in blood vessels observed in livers of G3 mice may be caused by hypoglycemia leading to starvation of host cells or perhaps to anoxia due to anemia caused by T. evansi 58 .Another explanation, suggested by Derakhshanfar et al. 59 who reported that dilation of focal areas and congestion in sinusoids caused by accumulation of lipids inside liver cells due to tissue hypoxia, resulted from anemia, and vascular damage.The present results are also in agreement with many authors who reported fatty degeneration in hepatocytes of rats infected with T. evansi 57,60,61 .
Results observed in mice treated with berenil are in agreement with Homeida et al. 62 who reported that berenil caused hemorrhage and degeneration in liver cells of camels infected with T. evansi.Adeyemi and Sulaiman 63 , also reported inflammation and congestion in hepatic and renal cells in rats infected with T. brucei and treated with berenil.These authors also mentioned elevation in alkaline phosphatase, alanine transaminase and aspartate transaminase from degenerative changes in hepatic cells.
Protective effects of hemolymph for histological structures of vital organs is consistent with Al-Otaibi et al. 64 who showed that methanolic extracts of natural products, such as from Lepidium sativum seeds, can reduce hepatotoxicity, and limit damage to hepatocytes.These extracts resulted in significant improvement and normalization of liver enzyme levels.
This study also showed that infection with T. evansi in control group mice and mice treated with berenil resulted in severe congestion in interstitial blood vessels, multifocal aggregation of mononuclear cells, degeneration in renal tubules, degeneration and infiltration in renal cells may be caused by invading trypanosomes 63 or perhaps by utilization of glucose and oxygen during parasite proliferation leading to deformities in kidney and liver 65 .Homeida et al. 62 also reported the same changes in the kidneys of camels infected with T. evansi.The present results are also in agreement with Mbaya et al. 66 who noticed various degrees of degenerative changes with cellular infiltrations in kidneys of gazelles infected with T. brucei and treated with diminazene aceturate at 3.5 mg/kg body weight.Results from G4 mice are supported by results of many authors who reported that natural products extracted from plants and insects and used as anti-trypanosomal drugs protect histological structures, and biochemical and enzyme systems of infected animals 34,45,67,68 .
Changes in spleen indicate a protective immune response correlated with inflammatory responses to infection.The inflammatory response may be the initial host response against protozoal infection or may be due to reduction in IL-10 levels 45,55 .Changes in splenic cells may be due to hypersensitivity to infection with T. evansi 58 or to a response of T. evansi to its toxic metabolites that results in varying degrees of anemic anoxia and splenic damage 69 .Study results are in agreement with many authors who report hemorrhage and focal necrosis in spleens of donkeys and rats 60,69 and with Morrison et al. 70 who reported the same changes in dogs infected with T. brucei.

Conclusion
This study marks the pioneering investigation into the chemical composition of Sarcophaga argyostoma larval hemolymph.Through chromatographic analysis, several valuable compounds were unveiled, showcasing potential for the refinement of therapeutic and pharmaceutical approaches in combatting parasitic and other related diseases.Notably, administration of Sarcophaga argyostoma larval hemolymph at a dosage of 0.5 ml/kg demonstrated a significant inhibition of T. evansi organisms in vivo, underscoring a robust trypanocidal effect.Moreover, hemolymph was found to modulate inflammatory mediators while exhibiting no adverse impact on immune system organs, such as the spleen.Furthermore, it conferred protective effects on vital organ structures, such as the liver and kidney, safeguarding them from damage induced by T. evansi infection.The administration of a prophylactic dose to potentially exposed animals within the region of risk showcases promising prospects in either preventing infection or bolstering the health of susceptible animals.

Figure 2 .Figure 3 .
Figure 2. Photomicrograph of liver sections.(A) and (B) liver histology of mice in the positive control group (G2); (A) showing diffuse and massive degenerative changes including granular cytoplasm (red arrow), nuclei are either pyknotic (blue arrow) or vesicular (black arrow).(B) Liver showing congested central vein (arrowhead) with massive, degenerated hepatocytes (arrows).(C) Liver histology of G3 (infected and treated with berenil) showing dilatation of the hepatoportal blood vessels (arrowhead) and diffuse vacuolation of the hepatic parenchyma (arrows).(D,E) Livers of extract-treated groups before and after infection (G4, G5) mice showing normal liver histology.(F) Normal hepatocytes in negative control mice (G1).H&E.

Table 2 .
The amino acid names identified in the hemolymph of Sarcophaga argyostoma larva using an amino acid analyzer.min.minute, PPM parts per million.